Autoantibodies to phospholipids (aPL) occur in certain patients with systemic lupus erythematosus, other autoimmune diseases, and the primary aPL syndrome. Their presence is associated with an increased risk of thrombosis, recurrent fetal loss, and thrombocytopenia. It is thought that these autoantibodies may exert a direct pathogenic effect by interfering with hemostatic reactions that occur on the surface of platelets and vascular endothelial cells. It has recently been reported that aPL do not recognize phospholipid alone, but rather a complex antigen comprised of phospholipid and the plasma protein beta2- glycoprotein I (beta2GPI). Interestingly, beta2GPI has certain relevant biological properties previously attributed to aPL, i.e., binding to anionic phospholipids and inhibition of prothrombinase activity. We have recently demonstrated that the ability of certain aPL to inhibit prothrombin activation i.e., their lupus anticoagulant activity, is dependent upon the presence of beta2GPI. It is hypothesized that antiphospholipid antibodies exert their pathogenic effects by enhancing the binding of beta2GPI to phospholipids, thereby inhibiting phospholipid-dependent reactions of the hemostatic system. The long-term objective of the proposed research is to define the mechanisms by which aPL may cause a thrombotic diathesis. The proposed studies will focus on the effect of aPL on the interactions of beta2GPI and phospholipids, both in binding studies and hemostatic reactions. The first aim is to characterize quantitatively the effect of aPL on the binding of beta2GPI to phospholipid membranes. Binding of beta2GPI alone will be quantitated by 90 degrees light scattering and fluorescence energy transfer. The effect of aPL on this binding will be determined using nonequilibrium binding assays and fluorescence anisotropy measurements. The second aim is to determine the mechanism by which aPL and beta2GPI inhibit the activation of prothrombin, and incorporate this information into a quantitative model of prothrombin activation. The third aim is to determine the effects of beta2GPI, in the presence or absence of aPL, on phospholipid-dependent reactions of the protein C pathway, i.e., the thrombin/thrombomodulin activation of protein C and the protein C/protein S inactivation of factors Va and VIIIa. Inhibition of the protein C pathway by beta2GPI and aPL on the interaction of B2GPI with phospholipid membranes may provide insight into the pathophysiology of the aPL syndrome and suggest novel therapeutic strategies.